Method for detecting a hormone or antihormone resistance in cancers

ABSTRACT

The invention concerns a method for detecting a hormone or antihormone resistance in cancers. The invention is intended for use in medicine, biology and the pharmaceutical industry. The aim of the invention is to consistently improve the use of antihormones in the treatment of cancer so that unnecessary treatment can be avoided as much as possible. The aim is to develop a method which can be used to identify the type of cancer before corresponding antihormone treatment is given. This aim is achieved by measuring the immune reactivity of the hormone receptors of cancers. The method as per the invention is characterized in that an antihormone receptor antibody, which is attached to a solid, is incubated with a prepared cytosol from the cancer tissue being investigated and, following removal of the liquid, is once again incubated with the addition of the respective hormone or antihormone, then a second enzyme-labelled antibody is added, the color reaction measured and the determined value for the immune reaction is compared with the control value which is obtained in the same way but without the respective hormone or antihormone being incubated.

This application is a national stage entry of PCT/DE97/01423, filed Jul.5, 1997.

BACKGROUND OF THE INVENTION

The invention concerns a method for detecting a hormone or antihormoneresistance in tumours. The invention is intended for use in medicine,biology and pharmaceutical industry.

Tumours in organs depending on hormones such as in the breast andovaries are tumour diseases met most frequently in women inindustrialized countries. Thus, in about 12% of the female populationbreast cancer is diagnosed, with the peak of sickening being between the4^(th) and 6^(th) decade of life. 3.5% of women die of it (Harris etal., New England J Med 327: 319-328, 1992).

Depending on the stage at which breast cancer is diagnosed the chancesof recovery are between 30 and 70%. As about ⅔ of the tumours detectedhave hormone receptors these patients are given hormone preparations asprimary medication. They are to competitively displace the naturalligand (oestrogen) from the receptor, thus affecting the growth of thetumours caused by hormones. Tamoxifen, a non-steroidal antioestrogen, ismost frequently used for treating mammary carcinomas with the adjuvantuse to prevent the development of metastases, yet also the prophylacticuse for patients with a family-connected risk (Harris et al., NewEngland J Med 327: 473-480, 1992).

It is known that about only half of the hormone receptor-positivepatients respond to a tamoxifen therapy. This raises questions relatingto the principle action of antioestrogens which, for the time being, arestill largely unclarified. Thus, Berthois et al. (Molecular and cellularEndocrinology 99: 259-268, 1994) described that the immune reactivity ofoestroqen receptors to an antibody will increase if oestradiol ortamoxifen are added in a cytosole test. The authors discuss a change ofconformation of the oestrogen receptor by an in vitro interaction withhormones and anti-hormones as a potential mechanism for an apparentincrease of the positions for the epitope binding.

Though tamoxifen is comparatively well-tolerated also in long-term useits possible cancerogenic potential has been discussed recently(Williams et al., Eur J Cancer Prev 1: 386-387, 1992).

SUMMARY OF THE INVENTION

The invention is aimed at improving the use of antihormones for thetherapy of tumours in order to avoid unnecessary therapies as far aspossible. Its aim is to develop a method which allows to predict theresponse of a tumour before applying a respective antihormone therapy.

Starting point of the invention is the surprising finding that theimmune reactivity of sensitive and at resistant tumours differs givenspecific conditions of reaction. If hormone receptors contained in thetumour are bound to an antibody, in the case of sensitive tumours, theirinmmune reactivity is increased by adding respective hormones orantihormones, however not in the case of resistant tumours. That means,the positions of epitope binding are apparently increased only in thecase of sensitive tumours.

This effect has been used for designing the method according to theinvention wherein an antihormone receptor antibody bound to a solidphase is incubated with cytosole prepared of the tumour tissue underinvestigation, the liquid is subsequently removed and adding theappropriate hormones or antihormones a repeated incubation i s performedand subsequently a second enzyme-labelled antibody is added, the colourreaction is measured and the value determined for the immune reaction iscompared with the control value determined in the same way, but withoutincubation with the appropriate hormones or antihormones.

In this description n we understand by hormones oestrogens (such as17β-oestradiol), progesterone or androgen, by antihormonesantioestroqens (such as tanoxifen or 4-hydroxy-tamoxifen),antiprogestins or antiandrogens (such as cyproteronacetate).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—diagram of the method of the present invention for detecting ahormone or antihormone resistance in cancers.

PREFERRED EMBODIMENTS OF THE INVENTION

The method is, in particular, described by means of the oestrogenexample. We use a commercial enzyme immunoassay which in clinicalroutine is used for determining hormone receptors. Polystyrene beadscovered by an antioestrogen receptor antibody will be incubated withcytosole prepared of the respective tumour tissues. After binding of theavailable oestrogen receptors to the primary antibody the liquid isremoved. After adding oestrogens or antioestrogens such as tamoxifen themixture is incubated repeatedly. Then, a second peroxydase-coupledantibody is added and its colour reaction is measured which allows adirect calculation of the available oestrogen receptor (FIG. 1).

The invention allows a reliable differentiation between sensitive andresistant tumours and thus an assessment of the chances of success of anantitumoural hormone therapy.

The invention is explained in greater detail hereinafter by an exampleof execution:

EXAMPLE

The results are based on experiments carried out with a hormonereceptor-positive mammary carcinoma (3366) established from patients'material as a xenotransplantation model on nude mice (H. Naundorf etal., J. cancer Res Clin Oncol 119: 35-40,1992). By treating this tumourwhich was originally extremely sensitive to tamoxifen in vivo withsuboptimal doses for a few years a resistant subline was produced.

The tumours of the mammary carcinoma 3366 are shock-frozen and preservedin liquid nitrogen until the test will be carried out. By means ofultra-sound the samples are homogenized at 2-8° C. 5 times in an icebath (10 sec.).

Cytosoles of the tamoxifen-sensitive and the tamoxifen-resistant linesare used in the enzyme immunoassay developed by the Abbott company. Thisenzyme immunoassay contains the antioestrogen receptor antibody D547 andthe peroxidase-coupled antibody H222 as second antibody the colourreaction of which is measured after adding o-phenyldiamine at 492 nm.The cytosole is prepared according to the instruction enclosed in theAbbott ER-EIA monoclonal kit and the protein content is set to 1-2mg/ml.

After incubation with antibody beads (D 547) the liquid is sucked off bymeans of a quick-wash facility (Abbott) in 8-12 ml of distilled water.Always 200 ml of the receptor hormone or antihornone are pipetted to thebeads as a double determination. 4-hydiroxytamoxifen, 17 β-estradiol,ICI 182,780 (Wakeling, J. Steroid Biochem Molec Biol 47: 107-114, 1991)and progesteron in concentrations between 10⁻⁴ and 10⁻¹⁰ molar are used.The most optimum results are obtained with 10⁻⁶ molar, aqueous solutionsprepared of a 10⁻⁴ molar ethanolic solution. In the control sample onlysolvent is used without adding hormones.

The residence time of the hormones or antihormones at the beads is 2hours, the temperature 37° C. (incubator).

Thereupon, the liquids are again sucked off by means of the quick-washand with the second antibody H222 being added. After reaction with theo-phenyldiamine hydrochloric acid solution the ER receptors are measuredaccording to the Abbott instruction in fmol/ml or extinction at 492 nm.

The results are summarised in the following table:

Immune reactivity (in percent) to antibody H222:

mammary carzinoma 3366 Treatment tamoxifen-sensitive tamoxifen-resistantwithout 100 100 4-hydroxytamoxifen 262 ± 53’ 140 ± 13 17b-oestradiol 243± 45’ 116 ± 17 ICI 182, 780   291 ± 100+ 123 ± 5  progesteron 91 ± 8  93 ± 10 (*significant as compared with the resistant line)

The results show that the immune reactivity increases significantly onlyin (anti)hormone-sensitive tumours whereas in resistant tumoursadditional antibody binding positions are not presented. Thus, this testmethod may be applied to distinguish between hormone and antihormonesensitive and resistant tumours.

Legend of FIG. 1

1—Incubation with tissue cytosoles, binding of the oestrogen receptor

2—Adding of oestrogens or antioestrogens

3—Incubation with a second antibody

4—Photometric measurement

5—Increased receptor value=sensitive tumour

6—Normal receptor value=resistant tumour

What is claimed is:
 1. A method for detecting hormone resistance orantihormone resistance in tumors, comprising the following steps, insequence: (a) obtaining cytosol from tumor tissue thereby producing aliquid sample, (b) contacting the cytosol with an antihormone receptorantibody to form a complex, (c) removing the liquid, (d) incubating thecomplex with a hormone or antihormone, (e) contacting the complex withan enzyme labeled antibody, and (f) measuring the degree of immunereaction as indicated by a resulting color change, and comparing it to acontrol, thereby determining whether the tumor tissue is eithersensitive or resistant to hormones or antihormones.
 2. The method ofclaim 1, wherein the antihormone receptor antibody is antioestrogenreceptor antibody.
 3. The method of claim 2, wherein hormone orantihormone is chosen from the group consisting of oestrogen, tamoxifenand antioestrogen.
 4. The method of claim 1, wherein the enzyme labeledantibody is peroxidase-coupled antibody.
 5. The method of claim 1,wherein step (d) is carried out with a 10⁻⁶ to 10⁻⁷ molar solution ofthe hormone or antihormone.